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Exploring miniature insect brains using micro-CT scanning techniques.

ABSTRACT: The capacity to explore soft tissue structures in detail is important in understanding animal physiology and how this determines features such as movement, behaviour and the impact of trauma on regular function. Here we use advances in micro-computed tomography (micro-CT) technology to explore the brain of an important insect pollinator and model organism, the bumblebee (Bombus terrestris). Here we present a method for accurate imaging and exploration of insect brains that keeps brain tissue free from trauma and in its natural stereo-geometry, and showcase our 3D reconstructions and analyses of 19 individual brains at high resolution. Development of this protocol allows relatively rapid and cost effective brain reconstructions, making it an accessible methodology to the wider scientific community. The protocol describes the necessary steps for sample preparation, tissue staining, micro-CT scanning and 3D reconstruction, followed by a method for image analysis using the freeware SPIERS. These image analysis methods describe how to virtually extract key composite structures from the insect brain, and we demonstrate the application and precision of this method by calculating structural volumes and investigating the allometric relationships between bumblebee brain structures.

SUBMITTER: Smith DB

PROVIDER: S-EPMC4764865 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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Exploring miniature insect brains using micro-CT scanning techniques.

Smith Dylan B DB Bernhardt Galina G Raine Nigel E NE Abel Richard L RL Sykes Dan D Ahmed Farah F Pedroso Inti I Gill Richard J RJ

Scientific reports 20160224

The capacity to explore soft tissue structures in detail is important in understanding animal physiology and how this determines features such as movement, behaviour and the impact of trauma on regular function. Here we use advances in micro-computed tomography (micro-CT) technology to explore the brain of an important insect pollinator and model organism, the bumblebee (Bombus terrestris). Here we present a method for accurate imaging and exploration of insect brains that keeps brain tissue fre ...[more]

PMID: 26908205

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Project description:BackgroundMicro-CT holds promising potential for phenotyping and histological purposes. However, few have clarified the difference in the neuroimaging quality between ex vivo and in vivo micro-CT scanners. In addition, no direct comparison has been made between micro-CT scans and standard microscopy. Furthermore, while the efficacy of various stains for yielding soft-tissue contrast in CT scans have been compared in other studies for embryos, staining protocols for larger samples have yet to be clarified. Lastly, post-acquisition processing for image enhancements have not been addressed.MethodsComparisons of postnatal rat brain micro-CT scans obtained through custom-built ex vivo and commercially available in vivo micro-CT scanners were made. Subsequently, the scanned rat brains were then H&E stained for microscopy. Neuroanatomy on micro-CT scanning and 4× microscopy of rat brain were compared. Diffusion and perfusion staining using iodine or PTA were trialled on adult and neonatal encapsulated rat brains. Different combinations of stain concentration and staining time were trialled. Post-acquisition denoising with NLM filter was completed using a modern General-Purpose Graphic Processing Unit (GPGPU) and custom code for prompt processing.ResultsEx vivo micro-CT scans of iodine-stained postnatal rat brains yields 3D images with details comparable to 4× H&E light micrographs. Neural features shown on ex vivo micro-CT scans were significantly more distinctive than those on in vivo micro-CT scans. Both ex vivo and in vivo micro-CT scans required diffusion staining through small craniotomy. Perfusion staining is ineffective. Iodine staining was more efficient than PTA in terms of time. Consistently, enhancement made by NLM denoising on in vivo micro-CT images were more pronounced than that on ex vivo micro-CT scans due to their difference in image signal-to-noise indexes.ConclusionsMicro-CT scanning is a powerful and versatile visualization tool available for qualitative and potential quantitative anatomical analysis. Simple diffusion staining via craniotomy with 1.5% iodine is an effective and minimal structural-invasive method for both in vivo and ex vivo micro-CT scanning for studying the microscopic morphology of neonatal and adult rat brains. Post-acquisition NLM filtering is an effective enhancement technique for in vivo micro-CT brain scans.

Project description:BackgroundChitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term "denatured chitin" calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished.Methodology/principal findingsA case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment.Conclusions/significanceThe technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an additional tool for research on CCD.

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Exploring miniature insect brains using micro-CT scanning techniques.

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Exploring miniature insect brains using micro-CT scanning techniques.

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